CH667593A5 - MEDICINAL PRODUCT FOR TREATING VIRUS INFECTIONS CONTAINING OLIGODEOXYNUCLEOTIDES AND / OR POLYDEOXYNUCLEOTIDES. - Google Patents

Description

DESCRIPTION The present invention relates to medicaments for the treatment of viral infections which contain either a single or a mixture of oligo- and / or polydeoxynucleotides of 9-100 nucleotides in the form of a (-) - DNA strand, which have one region hybridize an mRNA (messenger RNA) synthesized during virus propagation and a pharmaceutically acceptable excipient suitable for the treatment of virus infections. Medicaments of this type can be packaged, for example, as a solution for injection, as eye drops or as suppositories. The drugs mentioned can be used so that the amount of active ingredient administered is either 1 to 20,000 g per kg and day.

Furthermore, the invention relates to methods for the production of said medicinal products.

So far, chemically synthesized drugs and antibiotics have been used as antiviral agents to inhibit virus replication in the treatment of viral diseases. However, the antiviral spectrum of chemically synthesized drugs is relatively narrow and often such drugs have harmful side effects. On the other hand, the antibiotics used have the disadvantage that new antibiotics and efforts to reduce any harmful side effects of these antibiotics are constantly necessary, because over time the viruses become resistant and ultimately immune to the antibiotics used.

5 As part of the recent development of biotechnology, especially genetic engineering, the identification of virus structural genes has become a major area of research within the studies of microorganisms. In 1977 S.C. Inglis et al. the in vitro translation of cytoplasm-io matic RNA extracted from chicken embryo fibroblasts infected with influenza A virus in the cell-free wheat germ protein synthesis system with the result that the synthesis of virus-specific polypeptides involved the hybridized v-RNA segment correspond, is reduced (Viro-i5 logic 78, pages 522-536 (1977)). However, the experiments described in this publication were carried out in a cell-free system and have no relation to the investigation of drugs. Such structural gene identification is also described by B.M. Paterson et 20 al. (Proc. Nati. Acad. Sci. USA. 74, pages 4370-4374 (1977)). The experiments by Paterson et al. were also carried out in a cell-free system. In the two publications mentioned, a huge genetic structure such as RNA was used for the inhibition of protein synthesis. In the first case the RNA was extracted from influenza viruses, in the second case from the rabbit-ß-globin clone pßGl. Such a huge structure could not be expected to inhibit intracellular protein synthesis. In 1978, P.C. Zamecnik et al. an oligodeoxy nucleotide tridecamer complementary to the 3'- and 30 5'-terminal repetitive sequence of the Rous-Sarcoma virus (RSV) an effective inhibitor of protein synthesis based on viral RNA both in the cell-free wheat germ system (Proc. Nati. Acad Se. USA. 75, pages 285-288 (1978)) and 35 in an in vitro tissue culture system (Proc. Nati. Acad. Se. USA. 75, pages 280-284 (1978)) . In the first reference, the cell-free system is still used as described above. However, it is slightly improved in the use of DNA. Here a smaller DNA-40 molecule is used to inhibit the translation of mRNA. In the second publication, experiments are carried out in which in a tissue culture system is used to inhibit virus replication and the cell transformation of the tridecamer. The DNA used in this work 45 to inhibit replication and transformation was not selected from a specific coding region. In the same year, N.D. Hastie et al. That the hybridization of globin mRNA with its corresponding cDNA in a cell-50-free system specifically inhibits the translation of the mRNA (Proc. Nati. Acad. Sei USA 75, pages 1217-1221 (1978)). What is new in this work is only that a specific coding DNA region was selected for the inhibition of translation by means of hybridization of mRNA. However, the 55 experiments mentioned there were still carried out in a cell-free system. Since these publications in 1978, there has been a need for drug development based on the hypotheses mentioned. To date, however, there have been no publications describing the practical application of the theory mentioned in in vivo experiments. __

In 1983 a PCT application was published which relates to oligonucleotides as therapeutic agents and processes for their preparation (Molecular Biosystem 65 Inc., No. W083 / 01451 or EP-1 192 574 of Nov. 2, 1983). However, this application merely discloses that (-) - strand DNA from a certain coding region inhibited SV40-transformed cells.

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Although the in vivo expression is frequently used in Example 1 of this publication, the descriptions given there, taken from the overall context, only suggest in vitro experiments in cell culture systems. Even if such in vitro experiments are regarded as in vivo experiments, there are still no corresponding precise specifications for the conditions and are nothing more than a mere mention of the expectation of desired effects. However, this publication suggests the use of RNA-DNA hybridization techniques in a drug for the first time, but lacks detailed descriptions of its use as a drug. Therefore, the teaching given in the aforementioned PCT application is only a summary of the prior art described in the aforementioned publications.

There was therefore an urgent need for a completely new antiviral active ingredient for inhibiting virus multiplication in vivo in a living animal, the principle of which is based on the RNA-DNA hybridization technique and thus on the inhibition of the translation of viral mRNA.

It has been found that virus multiplication at the site of infection can be inhibited with an oligodeoxynucleotide or a polydeoxynucleotide, which hybridizes with an mRNA synthesized during virus multiplication, provided that the active substances mentioned reach the site of infection. By administering the oligo- and polydeoxynucleotides to animals with a virus infection, an antiviral agent is provided which inhibits the virus multiplication in vivo.

As described above, the hybridization of viral or globin RNA with the corresponding DNA to inhibit protein synthesis was known. However, the majority of the experiments in this regard were carried out in a cell-free system. Exceptions are Zamecnik's publication (Proc. Nati. Acad. Sei. USA 75, pages 280-284 (1978)) and the publication by Molecular Biosystems Inc. (PCT Application No. WO 83/01451). The latter describes the in vitro inhibition of virus replication in a cell culture system. However, Zamecnik's in vitro experiments do not use DNA from a specific coding region of the virus. The term “coding region” denotes the region of viral genes that plays an important role in the translation of viral proteins and that is transcribed into mRNA by the RNA polymerase. The Molecular Biosystems Inc. publication does not provide any precise information about the experimental conditions. The present invention is based on extensive research and experiments in in vitro as well as in vivo systems using specific oligo- or polydeoxynucleotides from a specific coding area. For the first time, these have made it possible to inhibit virus replication by means of the RNA-DNA hybridization technique to an extent that can be used practically for therapy. This makes it possible to use certain oligo- or polydeoxynucleotides as drugs to inhibit virus replication. The present invention thus differs from the prior art in two important points, namely in that DNA hybridizing with the virus mRNA was selected from a specific coding region and this selected deoxynucleotide is short and single-stranded, and also in that the above-mentioned active ingredients do extensive in vivo experiments have been developed up to practical applicability.

The invention accordingly relates to the medicament defined in claim 1 for the treatment of virus infections. An oligo- or polydeoxynucleotide consisting of 9-100 nucleotides in the form of a (-) - DNA strand or a mixture of these molecules is used as the active ingredient. These oligo- or polydeoxynucleotides are characterized in that they are complementary to a region of an mRNA synthesized during virus multiplication and can therefore hybridize with this mRNA.

It is described for the first time that protein synthesis, starting from a synthetic RNA as mRNA in an in vitro protein synthesis system, is inhibited by an oligodeoxynucleotide hybridizing with this synthetic RNA (cf. the example described below

25). Oligodeoxynucleotides. those that do not hybridize with the RNA did not inhibit protein synthesis in this system.

In addition, it was found that oligodeoxynucleotides corresponding to the (-) strand of the a-globin or β-globin DNA specifically inhibit the translation of the associated mRNA (cf. the example described below

26). Furthermore, it was found that the β-globin DNA specifically inhibits the translation of β-globin mRNA in this in vitro system. If DNA that did not hybridize to the a-globin mRNA was used in these experiments, the formation of a-globin was not inhibited. Similarly, the ß-globin synthesis was not inhibited by DNA that did not hybridize to the ß-globin mRNA. Furthermore, it was found that not only long-chain DNA, but also oligodeoxynucleotides in this system have a strongly inhibiting effect due to the hybridization.

Based on these experiments, further experiments with herpes simplex virus (HSV) infections were carried out. It was found that the number of syncytia (plaque-like holes created by HSV by fusion of infected cells) caused by HSV by administration of DNA or oligodeoxynucleotides. the hybridize with the immediately synthesized early mRNA of HSV on cells infected with this virus is significantly reduced.

In the experiments described above, CaCl; and calcium phosphate together with the DNA used to stimulate DNA uptake into the cell. In other experiments, however, it was found that the oligodeoxynucleotides can also be taken up without these calcium salts and that they inhibit the multiplication of HSV even under these conditions and suppress the corresponding cytopatic effects (cf. Example 7 described below).

From these experiments it became clear that (-) strand DNA inhibited the formation of syncytia by this virus by hybridization to an HSV-1 mRNA. On the basis of this observation, the DNA HSV-infected mice mentioned were administered. This has shown that such DNA can be used as an antiviral drug for the treatment of viral infections in vivo.

It is noteworthy here that at least one (-) - stranded DNA, which corresponds to the mRNA of a specific virus gene, is used as active ingredient in the medicament according to the invention. For this reason, the biosynthesis of cellular proteins, which is controlled by other mRNA molecules, is not influenced by the use of the DNA molecules described above.

There are two groups of viruses, namely DNA viruses (herpes simplex virus, adeno virus, vaccinia virus and others) and RNA viruses (influenza virus, Rhino virus and polio virus and others). RNA viruses carry either single-stranded RNA or double-stranded RNA as a genome. Single-stranded RNA viruses are classified into those, the (+) stranded RNA

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and into those that carry (-) strand RNA as a genome.

Viral proteins must always be synthesized by translating a viral mRNA with (+) strand polarity. Viruses with a double-stranded DNA genome start from their (-) strand DNA (+) - strand RNA (mRNA). As for double-stranded RNA viruses, (-) stranded RNA (mRNA) is formed from the (-) - RNA strand. The genome of (+) stranded RNA viruses functions as an mRNA, while the genome of (-) stranded RNA viruses acts as a template for the formation of (+) stranded RNA (mRNA).

Regardless of the mechanism of mRNA formation, the translation of a viral mRNA and damti inhibits the synthesis of viral proteins by a hybridizing (-) - strand DNA. The subject of the present invention can be used generally to inhibit virus replication. The use of herpes simplex virus (double-stranded DNA virus) and influenza virus (single-stranded RNA virus) described in the examples is only for illustration. The multiplication of viruses, such as the influenza virus, can thus be inhibited by the active compounds according to the invention. Adeno virus, leukemia virus, dengue virus. Rabies virus, hepatitis virus, measles virus, ence-phalitis virus. Parainfluenza virus, Rhino virus. Yellow fever virus or Epstein-Barr virus.

The active ingredients of the medicament according to the invention can thus be used generally for the prophylaxis or treatment of various virus infections of animals, humans and plants. The corresponding drugs are therefore very useful for various applications.

Due to the principle of action of the active ingredients, the multiplication of RNA or DNA viruses and the occurrence of the corresponding cytopathic effects are blocked without influencing the host cells. In addition, the use of the active compounds according to the invention can prevent infection of host cells which have not yet been infected. It could be shown that the sole use of oligodeoxynucleotides hybridizing with the immediately synthesized early mRNA of the herpes simplex virus had no effects on normal, uninfected baby hamster kidney cells (BHK cells) or effects on mice. It was concluded that the oligodeoxynucleotides have no dangerous effects on normal cells and animals, nor that they affect the growth rate of these cells in any way.

The (-) - stranded oligo- and or polydeoxynucleotides used in the present invention can be obtained by enzymatic hydrolysis of DNA, denaturation and subsequent separation by affinity chromatography. On the other hand, synthetic (-) stranded oligodeoxynucleotides can also be used. The (-) stranded deoxynucleotides used in the experiments can thus be obtained using various methods.

The reserve transcriptase can be used to clone an RNA virus gene. In some cases, viral DNA can be used that is present in the infected cells and is derived from the original RNA virus genome. For example, the À phage (Charon phage) or the plasmid pBR 322 can be used as the cloning vector. To obtain a single-stranded (-) - strand DNA. the M 13 phage is used as a vector. Cloned fragments from the coding region of the entire virus genome can be used. However, the use of oligo- or polydeoxynucleotides which correspond to the immediately expressed early virus genes is preferred for the inhibition of virus replication and the associated cytopathic effects. A recombinant M 13 phage containing (-) stranded virus DNA or a recombinant DNA vector containing double stranded virus 5 DNA can be grown by growing large amounts of E. coli bacteria carrying these vectors or are infected with said M 13 phage, the vectors carrying the viral genes were isolated and cut with restriction endonucleases io to obtain the viral genes. The mixture of the excised virus genes and the vector DNA was then subjected to agarose gel electrophoresis or a Sephadex column chromatography to separate the virus gene from the vector DNA. The DNA molecules thus obtained were shortened by partial digestion with DNA restriction endonuclease or by ultrasound treatment. Virus DNA fragments with a chain length of 9 to 100 nucleotides were isolated using gel electrophoresis or Sephadex chromatography.

20 It is noted that only the (-) strand of virus DNA is effective in inhibiting virus protein synthesis. DNA from vectors such as pBR 322 or the X phage or from the replicative form of the M 13 phage is double-stranded DNA. Therefore, this double-stranded DNA must first be converted into single-stranded DNA before the (-) strand DNA is isolated. Various methods can be used for this purpose. For example, DNA can be denatured by heating to 100 C for 10 minutes and then rapidly cooling.

The (-) strand can be isolated from a mixture of the separated (-) - stranded and (+) stranded DNA, for example by affinity chromatography. For this purpose, the (+) DNA strand 35 is first produced by cloning in the M 13 phage. This is subsequently conjugated to a support for use in chromatography. A mixture of (-) - and (+) - strand DNA is then chromatographed on a column containing this conjugate. The (-) strand of DNA is bound to this column 40 while the (+) strand is running. The bound (-) - DNA strand is then eluted from the column.

In another method, the (-) - strand DNA is obtained in the form of oligo- or polydeoxynucleotides by chemical synthesis.

For example, one of the immediately experimented early genes of the double-stranded DNA-carrying herpes simplex virus, Vmw 175, has the (+) stranded DNA sequence 5'-

ATG.GCG.TCG.GAG) and the (-) - strand DNA

50 sequence (3'-TAC.CGC.AGC.CTC). Only the (-) -

Strand DNA with the sequence starting at TAC can hybridize with an mRNA synthesized starting from Vmw 175. For this reason, a DNA fragment is selected for the oligodeoxynucleotide synthesis which is identical to a partial structure of the (-) DNA strand. The influenza virus genome contains the hemagglutinin gene and NS (non-structure) genes. The hemagglutinin gene of different serotypes of the influenza virus differs in the RNA sequence. Therefore, according to the invention, a single-60-stranded DNA is preferred which hybridizes with mRNA of the NS genes. The use of (-) - stranded DNA, which hybridizes with mRNA of the NS genes, is therefore preferred according to the invention compared to the use of (-) - stranded DNA, which hybridizes with mRNA of the hemagglutinin gene, before-65 because the hemagglutinin gene very often mutates. A (-) strand DNA that hybridizes with mRNA of the hemagglutinin gene of a particular influenza virus strain is therefore not with mRNA of the hemagglutinin gene

Cross hybridize another serotype of the influenza virus.

A further explanation of the influenza virus gene NS-1 can be found in the publication by Lamb et al., (Cell 21, page 47 (1980)). The DNA sequence with an mRNA

of the NS-1 gene is 5'-ACT.TGA.CAC.

AGT.GTT.GGA.ATC.CAT -3 '. Part of this sequence or all of the sequence is therefore selected and synthesized.

The active ingredients of the medicament according to the invention can also be used against viruses, such as the Rous Sarcoma virus, which play an important role in the field of animal husbandry. For example, inhibition of the expression of one of the genes gag, pol or env should make it possible to inhibit the multiplication of this virus. If, for example, the expression of the gag gene is to be inhibited, a hybridizing DNA molecule, an oligonucleotide or a polydeoxynucleotide is used which contains part of the sequence 5'-AAT.CAT.CTT.TAT.GAC.GGC.TTC -3 'contains, selected and synthesized. The pl9 RNA sequence mentioned is part of the gag gene.

It follows from the above explanations that, in principle, for the inhibition of virus multiplication by means of a single-stranded (-) strand DNA molecule, at least one such molecule must be used that hybridizes with the mRNA of the virus.

In general, the term "oligodeoxynucleotide" refers to a molecule that contains a smaller number of deoxynucleotide units, while the term "polydeoxynucleotide" describes a molecule that contains a larger number of deoxynucleotide units. For example, in the case of oligodeoxynucleotides, the number of nucleotide units in this invention is up to 25. Therefore, deoxynucleotides containing more than 25 deoxynucleotide units can be referred to as "polydeoxynucleotides" in this invention. The medicaments according to the invention for the treatment of viral infections contain at least one oligo- and / or polydeoxynucleotide from 9-100 nucleotides in the form of a (-) - DNA strand together with a suitable liquid vehicle or excipient or optionally at least one additive. They are produced in processes known per se, for example as liquid preparations, injection preparations or suppositories. If appropriate, the oligo- and / or polydeoxynucleotides can be used either individually or in combination with other pharmacologically active substances. It goes without saying that these other pharmacologically active substances should not conflict with the antiviral activity of the oligo- and / or polydeoxynucleotides mentioned.

The oligo- and / or polydeoxynucleotides can optionally be made up into externally applicable preparations, such as creams, ointments, drops or plasters. For this purpose, the oligo- and / or polydeoxynucleotides are mixed with a suitable pharmacologically inert vehicle or excipient.

The liquid carriers or excipients and any additives used, which are used according to the invention as an additive to the antiviral active ingredients, are known and commercially available.

Examples of the liquid excipients and excipients used in the antivirally active drugs are distilled water for injections, physiological saline, an aqueous dextrose solution, vegetable oils for injections, propylene glycol, polyethylene glycol and benzyl alcohol (for injection and liquid preparations); Vaseline, vegetable oils, animal fats and poly-ethylene glycol (for externally applicable preparations); isoto5 667 593

nically active substances, substances which improve solubility properties, stabilizers, dyes, antiseptics, lubricants and similar additives. In the case of the injection preparations, the active ingredient is preferably dissolved in a sterilized liquid vehicle. It goes without saying that the various liquid vehicles, excipients and auxiliaries mentioned above should neither inhibit nor interfere with the antiviral action of the oligo- and / or polydioxynucleotides.

The oligo- and / or polydeoxynucleotides are processed together with the aforementioned vehicles or excipients and optionally together with the auxiliaries described above in a manner known per se. Injection preparations and suppositories can contain 1 to 10 mg of the oligo- and / or polydeoxynucleotides per ampoule or capsule. In human patients, the daily dose is about 0.1 to 1000 mg, preferably 1 to 100 mg (from 10 to 20 µg / kg to 1000 to 2000 ng / kg body weight). However, the particular dose for certain patients depends on a number of factors, for example on the effectiveness of the particular oligo and / or polydeoxynucleotide used, on the age, on the weight, on the general state of health, on the sex, on the diet, on the time and the type of application, 25 the rate of breakdown, the combination with other medicines used in connection with it, and the severity of the disease in question against which this therapy is used.

The antiviral drugs according to the invention are applied intravenously or applied directly to the body part of the patient infected with the virus, so that the oligo- and / or polydeoxynucleotides reach the infected body parts in a suitable amount.

In order to facilitate an understanding of the principles of the present invention, a brief summary of the properties of "oligo- and / or polynucleotides of 9-100 nucleotides in the form of a (-) - DNA strand" is given below:

1. Properties and structure of DNA: A DNA suitable for the present invention has to hybridize with an mRNA which is generated during virus multiplication, as can be seen in FIG. 5.

DNA (a) that contains a segment that fully hybridizes with the mRNA and DNA (b) that contains a segment 45 that partially hybridizes with the mRNA is suitable for the purposes of the present invention. This characteristic is represented by the definition “in whole or in part with a DNA that hybridizes during the multiplication of viruses”.

2. The DNA region selected for the hybridization with the mRNA according to the present invention must lie within the segment which hybridizes completely or at least partially with the mRNA generated during virus multiplication, as can be seen in FIG. 6. 55 In the case of (c), a region (i) or (ii) is selected for the hybridization which hybridizes completely with the mRNA and thus meets the requirements. In the case of (d), on the other hand, a region (iii) is selected for the hybridization which lies outside the segment which fully or partially hybridizes with the mRNA. The latter does not meet the relevant requirements.

3. It follows that the oligo- or polydeoxynucleotides (A) or (B) used according to the invention for hybridization to 65 mRNA and the inhibition of the translation of mRNA caused thereby, i.e. the protein synthesis, in its DNA sequence must be identical to the region of a DNA (c) hybridizing with the mRNA (for example 5′-

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... .ACT.TGA.CAC ... .ATC.CAT -3 '). For the better

Please refer to Fig. 7 for understanding.

Preferably the deoxynucleotide chain (A) or (B) is as short as possible because the synthesis of a short deoxynucleotide is easy and economical. The use of certain eicosamers (i.e., oligonucleotides consisting of 20 nucleotide units) to obtain stable hybridization to mRNA is described in the examples of the present invention.

The advantageous effect of the active ingredients of the medicament according to the invention can be seen from the diagrams in FIGS. 1 to 4 and the following examples.

Figure 1 is a graphical representation of the results of experiments showing the inhibitory effects of oligo deoxynucleotide 20A on the cytophatic effects of HSV-1.

Figure 2 is a graphical representation of the results of experiments showing the inhibitory effects of oligodeoxynucleotide 20A on virus replication.

Figure 3 is a graphical representation of the effect of a particular combination of oligodeoxynucleotides in inhibiting virus replication and the pathogenic changes thereby caused when compared to the use of a single oligodeoxynucleotide.

Figure 4 is a graphical representation of the results of experiments performed to demonstrate the in vivo activity of the antiviral agents.

Examples 1 to 5 relate to the production of oligo- and polydeoxynucleotides.

example 1

Herpes simplex virus DNA was isolated by phenol-chloroform extraction. The isolated DNA was digested with the restriction enzyme BamHI.

The digested DNA was then mixed with pBR 322 plasmid DNA previously treated with BamHI in the presence of T 4 phage ligase.

The resulting mixture containing ligated plasmids was administered to E. coli bacteria and the bacteria containing the recombinant plasmid were selected by incubation on ampicillin-enriched agar plates on which they formed colonies and by examination for tetracycline sensitivity. The bacteria containing herpes simples virus DNA were selected from the tetracycline-sensitive colonies by the filter hybridization method with 32P-labeled herpes virus DNA as the probe molecule. To contain DNA of the recombinant plasmid, 2 x IO6-IO7 E. coli cells containing the recombinant plasmid were placed in 2 ml TSB (tryptose soy broth).

The inoculated medium was shaken at 37 C in a 20 ml sample tube for about 15 hours until the optical density (OD) of the culture medium at 540 nm reached a value of 0.5 (OD54o = 0.5). At this optical density, chloramphenicol was added to a final concentration of 100ng ml and the incubation continued for a further 15 hours. The bacterial suspension was then centrifuged and the pellet was resuspended in a buffer containing 25% sucrose and 50 mM Tris-HCl (pH 8.0). The suspension was left to stand at 0 ° C. for 5 minutes. 4 ml of Triton X 100 were added to this suspension and the resulting viscous solution was centrifuged for 30 minutes at 0 ° C. and 30,000 g. To the supernatant of this solution (8 ml) was added 8 g CsCl and 1 ml of an ethidium bromide solution (5 h of ethidium bromide / ml) were added, the mixture was further centrifuged at 100,000 g for 48 hours, the crude plasmid fraction obtained by this centrifugation was mixed with 2 volumes of isopropyl alcohol and a few minutes The top layer was removed and the DNA containing solution was dialyzed against 0.1 molar Tris-HCl and 10 mM EDTA (pH 8.0) overnight. The dialyzed solution was concentrated and the 200 µg DNA solution was concentrated with an excess of the restriction enzyme BamHI, the cleaved DNA was separated by agarose gel electrophoresis and the herpes simplex virus DNA was isolated from the io gel by electroelution. Furthermore, the DNA was partially digested with DNase, heated to 100 ° C. for 10 minutes and then rapidly cooled. In this way, DNA with a chain length of 9 to 100 oligodeoxynucleotides was obtained.

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Example 2

The replicative form of the M 13 phage DNA (double-stranded DNA) was cut with BamHI. The M 13 DNA thus cleaved was previously mixed with BamHI-cleaved 20 and herpes simplex virus DNA isolated as described above. The ligation was carried out using T 4 phage ligase. E. coli were transfected with the ligated DNA in the presence of CaCl :. The transfected bacteria were plated on agar plates. A top agar was used which contained IPTG (isopropyl-β-D-thioglactopyranoside) and X-gal (an indicator). Herpes simplex virus DNA-containing recombinant M 13 phages form colorless plaques within 24 hours. M 13 phages without insertion do not form any plaques. The colorless plaques were therefore selected. To select recombinant M 13 phages containing the (-) strand HSV DNA, the phages were checked for their ability to hybridize with labeled (-t -) strand HSV DNA that was chemically synthesized . In this way, M 13-Pha-35 genes were selected which contain part of the (-) - stranded DNA from HSV. The desired recombinant M 13 phages were grown and single stranded DNA was isolated from these phages. For further purification of the single-stranded HSV DNA, the M 13 phage DNA can be removed from the mixture with the aid of a cleavage with a restriction enzyme in the presence of certain oligodeoxynucleotides. On the other hand, the DNA mixture can be partially cleaved with DNase without purification and oligodeoxynucleotides with a chain length between 9 and 100 can be isolated.

Example 3

A DNA synthesis device (Applied Biosystem so Company) was used to synthesize (-) strand DNA of the immediately expressed early gene Vmw 175 or ICP 4 of the herpes simplex virus. In this example the DNA sequence 3'-

CGC.AGC.CTC.TTG.TTC.GTC.GC-5 ', which hybridizes 55 with the mRNA of Vmw 175. This deoxynu-cleotide is an eicosamer and is simply referred to as 20 A below.

60 Example 4

A DNA synthesizer (Applied Biosystem Company) was used to synthesize (-) strand DNA of the immediately expressed early gene Vmw 12 of the herpes simplex virus. In this example, the synthesized DNA sequence was 3'-GCA.CCC.GGG.ACC-TTT-ACC-GC-5 '. This hybridizes with mRNA from Vmw 12. This deoxynucleotide is an eicosamer and is simply referred to as 20 B below.

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Example 5

A DNA synthesizer (Applied Biosystem Company) was used to synthesize a (-) strand DNA of the NS-1 gene of the influenza virus. In this example, the DNA sequence synthesized was 3'-CTA.AGT.TTG.TGA.CAC.AGT.TCA-5 '. This hybridizes with mRNA from NS-1. This deoxynucleotide is a heneicosamer and is simply referred to as 20 C below.

Example 6

This example relates to an attempt to confirm that the oligodeoxynucleotide inhibits the synthesis of virus protein in vitro.

Table I shows the inhibitory effect of 20 A on Vmw 175 formation and the corresponding cytopatic effects (syncytium formation) of HSV 1

Table I

20 A hole Relative amounts Relative expression of the cytopathic effect of Vmw 175

(Syncytium)

1 1

33.6 (ig 0.48 0.35

BHK cells (1.5 x 105) were plated in each well (1.5 cm in diameter) of a Limbro plate and MEM enriched with 10% calf serum was added. The cells were then incubated in a 5 percent CO; atmosphere at 36 ° C. Subsequently, the cells with

5 moi HSV-1 infected in the presence or absence of 20 A. The medium was removed after 5 hours. The cells were then contacted with 35S methionine (100 | iCi / nl) for 1 hour. Subsequently, 20 A was broken down and the proteins were analyzed by polyacrylamide gel electrophoresis with subsequent autoradiography. Identical conditions to those described above were used to evaluate the relative cytopathic effect, except that the number of syncytia was determined 13 hours after infection. No decrease in cellular protein synthesis was observed under these conditions.

Example 7

This example relates to an attempt to confirm that the oligodeoxynucleotide is taken up by cells.

This example shows that the single-stranded oligodeoxynucleotide 20 A actually penetrates cultured cells. BHK cells (baby hamster kidney cells; 4.4 x 105 cells) were infected with 7.6 x IO4 PFU from HSV-1 / ml. Control cells were not infected, but the same amount of culture medium was added. The cells were incubated for 3 hours in a COi incubator at 37 ° C. After the incubation, the cells were exposed to 7.66 pmol of 32P-labeled 20 A. After treatment with the labeled 20 A, the culture medium was removed and the cells were washed twice with 0.3 ml of PBS.

The cells were then left to stand at 36 ° C. for 30 minutes in a solution containing 0.1 M NaCl, 33 | iM ZnCb, 3360 units of Nuclease S1 and 33.3 mM sodium acetate buffer (pH 4.5).

The cells were then washed twice with the buffer described above, but without Nuclease S1. The washed cells were in 0.3 ml of a solution with 10 mM EDTA, 0.6% sodium dodecyl sulfate and

10 mM Tris-HCl buffer (pH 7.5) digested. At this point, TCA-insoluble, nuclease SI-sensitive radioactive 20 A was measured. It was found that at least 4.68 x 103 20 A molecules per cell had been taken up during the 8 hour treatment period. The actual amount is likely to be 10 times higher than this because the terminal 32P phosphates are rapidly hydrolyzed.

The following Example 8 illustrates that 20 A has an inhibitory effect on virus replication.

Example 8

About 1.5 x 105 BHK cells were applied per well (1.5 cm in diameter) of a Limbro plate. These cells were incubated in 5% CO: 48 hours at 36 C and infected with HSV-1 (7.6 x 104 PFU ml) in 160 μl MEM. The infection was 3 hours at 36 C in 5% CO; executed. After infection, the infected cells were treated with 20 A. After a treatment period of 8 hours, the medium was exchanged for normal MEM with 10% calf serum. The culture medium was harvested 15 hours after the infection and the virus titers were determined. The results are summarized in Table II below.

Table II

Lot of 20 A holes Relative virus amount

0 100

0.112 Hg 45

0.224 | ig 35

Examples 9 to 13 relate to experiments confirming that the oligodeoxynucleotide inhibits the destruction of animal cells by the virus in vitro.

Example 9

(a) Approximately 1.5 x 105 BHK cells were placed in each well (1.5 cm in diameter) of a Limbro plate for cell culture. The cells were kept at 36 C in 5% CO for 47 hours; incubated and then infected with 160 yr 1 HSV-1 (7.6 x 104 PFU ml) at 36 C for 3 hours. A complex of calcium phosphate and 20 A in various amounts was added to these infected cells. The calcium concentration of the media was between 1.36 mmol and

24.8 mmol, depending on the 20 A concentration. The complex was developed as described by Ruddle et al. (Loiter et al., Proc. Nat. Acad. Sci. 79, p. 422 (1982)). The infected cells were treated with 20 A for 8 hours. The culture medium was then removed and replaced with a normal culture medium without 20 A. The cells were then grown for a further 13 hours.

(b) The control cells were treated in the same manner except for the addition of 20 A (Ca phosphate control).

(c) As a further control, an infected culture was prepared which was neither treated with Ca ions nor with 20 A (virus control). The tissue culture cells (a) prepared as described above. (b) and (c) were fixed, stained and the number of syncytia was determined.

The result of this experiment is shown in Figure 1. The X-axis (abscissa) indicates the amount of DNA as the added quantity in ig per well (ie “Hg DNA well”). The Y-axis (ordinate) indicates the number of syncytia formed in a cell culture in comparison a value. which is obtained in experiment (c) («virus control»)

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(i.e., "° or number of syncytia"), the latter value always being assumed to be 100. The curve (a) (- • -) shows the results of the experiment (a), the curve

(b) (O) shows the results of experiment (b).

As shown in Figure 1, the formation of syncytia was remarkably inhibited by 20 A. Calcium phosphate or calcium chloride did not affect the number of syncytia.

Example 10

(a) Approximately 1.5 x 10 BHK cells were drilled per well

(1.5 cm in diameter) a Limbro plate. The cells were incubated for 47 hours at 36 C in a 5% CO: atmosphere. 160 μl HSV-1 (7.6 × 104 PFU ml) were added to these cells. The infection was carried out at 36 C for 3 hours. Various amounts of 20 A in the form of a calcium chloride complex were added to cells infected in this way, so that the final Ca ion concentration was 4.55 mM. The treatment was continued for 8 hours. The cells were then incubated for an additional 14 hours.

(b) In addition, a culture identical to culture (a) described above was prepared, but using an oligodeoxv nucleotide. which was not related to the HSV-1 sequence 5 -CAC.GAC.AGA.GGG.CGA.-3 '. and incubated with it. All other conditions were identical to experiment (a).

(c) A similar culture was prepared, but without 20 A, and incubated under the above conditions. This is referred to below as "virus control".

The cells of experiments (a), (b) and (c) described above were fixed, stained and the number of syncytia was determined. The results of this experiment are shown in Figure 2. The X-axis (abscissa) indicates the amount of DNA added per well in jag (ie “jag DNA well”) and the Y-axis (ordinate) indicates the number of syncytia formed in a cell culture compared to a value , which was obtained in experiment (c) («virus control») (ie «% number of syncytia»), the latter value always being assumed to be 100. Curve (a) (- • -) shows the results of experiment (a), curve (b) (O) shows the results of experiment (b).

As shown in Figure 2, 20 A has an inhibitory effect on the number of syncytia formed by HSV-1. From this experiment it can be concluded that 20 A inhibits the formation of syncviene in the presence of 4.55 mM calcium ions. Oligodeoxynucleotides that are not related to the known DNA sequence of the herpes simplex virus had no inhibitory effect. It became clear from this experiment that the (-) strand DNA of the herpes simplex virus specifically inhibits the cytopathic effects of the herpes simplex virus.

Example 11

In Example 9, the effect achieved is shown using the synthetic 20 A eicosamer, which is related to the (-) strand of the HSV DNA. This experiment relates to the effect of cloned herpes sim plex virus DN A. It was also possible to inhibit syncytium formation by HSV-1 (1.45 megadalton.) Using a recombinant pBR 322 vector containing HSV-1 DNA , obtained by cleaving the HSV-1 DNA with BamHI and incorporating the fragments into pBR 322). In this experiment, the HSV-1 DNA was not excised from the recombinant plasmid. The denatured and partially cleaved recombinant pBR 322 DNA containing HSV-1 DNA was introduced into each well of a Limbro plate (0.16 (ig) as described in Example 9. The effect was evaluated as described in Example 9 and inhibition of syncytia formation was observed. pBR 322 s DNA was used as a control for this experiment. No inhibition was observed in this control experiment.

Example 12

This example shows a synergistic effect of an io mixture of oligodeoxynucleotides.

About 1.5 x 105 BHK cells were placed in each well (1.5 cm in diameter) of a Limbro plate. The cells were kept at 36 C in 5% CO for 47 to 48 hours; incubated and infected with HSV-1 (7.6 x 104 PFU / ml) in 160 μl MEM 15. The infection was 3 hours at 36 C in 5% CO; executed. After the infection, 20 A was dissolved alone or in a mixture with 20 B (224: 1) in a solvent and 0.3 ml of this solution was used in the presence of 4.55 mM calcium ions to treat the cells. The treatment of the cells with 20 A and 20 B was carried out for 8 hours.

Other cells were treated as described above, but 20 A and 20 B were not added. This attempt is referred to below as "virus control". 25 The cells were fixed, stained and the number of syncytia was determined. The results of these experiments are shown in Figure 3. The X axis (abscissa) stands for the amount of DNA added to each well in jag and the Y axis (ordinate) indicates the number of syncytia formed in a cell culture compared to a value that was Virus control ”was obtained (ie“% number of syncytia ”), the latter value always being assumed to be 100. The curve labeled "20 A" (- O -) shows the results of the experiment with 20 A alone, the curve labeled "20 A + 20 B (224: 1)"

(- # -)

shows the results of the experiments with 20 A and 20 B in a mixing ratio of 224: 1. As shown in FIG. 3, 40 the observed in vitro effect was stronger when using a mixture of 20 A and 20 B than when using 20 A alone. From this result it is concluded

that the joint use of (-) - stranded DNA molecules corresponds to the different regions of the DNA sequence 45 (-) stranded DNA from herpes simplex virus significantly increases the effectiveness as an antiviral active substance.

Example 13

50 This example shows the effect of (-) strand OHgo deoxynucleotides of NS-1 on the cytopathic effect caused by influenza viruses. The results of the investigation are summarized in Table III, the experimental procedure is described in more detail below.

Table III

Oligodeoxynucleotide Relative value based on

((ig ml) the number of survivors

60 cells

0 0.21

0.02 0.49

1.30 0.49

65 5.00 0.49

Pig kidney kitazato (SKK) cells (1.5 x 105 / 0.1 ml) were in 5% fetal calf serum containing

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MEM grown and placed in a hole (0.5 cm in diameter) of a microtiter plate. These cells were incubated for 24 hours at 35.5 ° C in a 5 percent CO2 atmosphere in the medium mentioned above. The medium was then removed and 100 μl of medium containing various amounts of the 20 C oligodeoxynucleotide was added to each well. The cells were then incubated for a further 24 hours in a 5% CO 2 atmosphere. The solution was then removed and 50 ml of medium containing 10 TCID50 units of influenza A virus were added to each well. Furthermore, 50 μl of the culture medium containing different amounts of 20 ° C. were added to each well. The cells were then further 48 hours The medium was then removed and 100 ml of Hank's solution containing 0.1% neutral red were added to each well and the cells were incubated for 1 hour at 35.5 ° C. in a 5% CO2 atmosphere was removed and the cells were washed with 10 × 80% ethanol and then 50 ml of 0.1 N HCl were added The relative number of surviving cells was determined by measuring the optical absorption at 577 nm using ELISA. Red while the non-surviving cells did not take a neutral red, so the higher optical density corresponds to a higher number of surviving cells.

Examples 14 to 20 below show the in vivo action of the oligodeoxynucleotides as an antiviral active ingredient.

Example 14

The intracerebral cavities of 3-week-old BALB / c mice were injected approximately 1 x 104 PFU HSV-1 (F strain) in 30jj.1 MEM. The virus suspension contained different amounts of 20 A. In addition, 200 μl of a MEM solution containing 1% penicillin, strep-tomycin and 2 mM glutamic acid and various amounts of 20 A were injected into the intraperitoneal cavities daily for 3 days, 1 Day after the injection of HSV-1 was started. For the 7th day, the mortality rate was calculated in percent. The result of the experiment is shown in Figure 4. The X axis (abscissa) indicates the amount of DNA injected into each mouse in [ig, i.e. "[Ig DNA / mouse" and the Y-axis (ordinate) indicate the mortality rate in% compared to that obtained in a control experiment in which the mice were injected with the same solution but without 20 A , the control mortality rate was always assumed to be 100. Figure 4 shows that the mortality rate was reduced by 20 À depending on the amount used.

The drawing gives the mortality rate as a percentage in comparison with the control group of mice that did not receive 20 A at all.

Example 15

About 1 x 104 PFU HSV-1 (F strain) in 30] ii MEM solution containing different amounts of 20 A was injected into the intracerebral cavities of 3-week-old BALB / c mice. In addition, 200 μl of a solution containing different amounts of 20 A, 1% penicillin and streptomycin and 2 mM glutamic acid were injected intraperitoneally once a day. It started 1 day after the injection of HSV-1.

Treatment was continued for 6 consecutive days. The mortality of the mice was determined 64 to 68 hours after the HSV-1 injection. The results are summarized in Table IV.

Table IV

Dose mortality in the mice

5/19

0.224 (ig / mouse 0/9

2.240 (ig / mouse 0/10

As can be seen from the table, 5 of a total of 19 mice died in the control group when no 20 A was administered. On the other hand, when 0.224 (ig 20 A or 2.240 ig 20 A was administered, no mice died at this time (a total of 19 mice were treated with both concentrations).

Example 16

Approximately 5 x 104 PFU HSV-1 (F strain) in 30 ml of a MEM solution containing different amounts of 20 A were injected into the intracerebral cavities of 3 week old BALB / c mice. The mortality rate of the mice was examined 64 to 68 hours or 71 to 78 hours after the injection. The results are summarized in Table V:

Table V

Dose mortality between mortality between 71

64 and 68 hours and 78 hours

- • 3/10 3/10

I, 12 ng / mouse 0/10 2/10

II, 2 ng / mouse 0/10 0/10

From this table it can be seen that in the control group which did not contain 20 A, 3 mice out of a total of 10 mice died. On the other hand, the mortality of the mice that received the virus in the presence of 20 A was greatly reduced.

Example 17

About 5 x 104 PFU HSV-1 (F strain) in 30 μl MEM solution containing certain amounts of 20 A was injected into the intracerebral cavities of 3 week old BALB / c mice. In addition, from the day after the injection, 30 ml of a MEM solution containing various amounts of 20 A, 1% penicillin and streptomycin and 2 mM glutamic acid were injected into the intracerebral cavities.

Treatment was continued for 2 days and the mortality of the mice was examined at various times after the virus was injected. The results are summarized in Table VI.

Table VI

mortality

Dose between 64 between 71 between 88

and 68 hours and 78 hours and 92 hours

1/10 3/10 7/10

U2 | xg /

Mouse 0/20 2/20 5/20

As can be seen from this table, the mortality was significantly higher in the control mice that did not receive 20 A than in the mice that were treated with 20 A. Although the effectiveness of 20 A appears to decrease at 88 to 92 hours after injection, the mortality in the control mice was even higher even under these conditions.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

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10th

Example 18

Approximately 1 x 10 "PFU HSV-1 (F strain) in 200 x 1 MEM solution containing 1.12 (ig 20 A) were injected into the peritoneal cavities of 3-week-old BALB / c mice. In addition, approximately 3 or 2 hours before the HSV-1 injection and 1 day after the injection of HSV-1 200 (in a MEM solution containing different amounts of 20 A, injected intraperitoneally into the infected mice. The intraperitoneal injection of 20 A was 2 days Mortality was assessed at 211 and 259 hours after HSV-1 injection and the results are summarized in Table VII.

Table VII

Dose mortality at mortality at

211 hours 259 hours

1/10 2/10

0.244 (ig mouse 0/10 0/10

As can be seen from this table, one out of 10 mice (at 211 hours after injection) or two out of a total of 10 mice (at 259 hours after injection) died when 20 A was not administered. On the other hand, no mice that received 0.224% 20 A died under the same experimental conditions.

Example 19

The corneal 3-week-old BALB / c mice were injured by scratching 5 times with a sharp injection needle. 30 (il MEM solution containing 1% penicillin, streptomycin, 2 mM glutamic acid, 5% calf serum and 3 x 104 PFU HSV-1 (F strain) were applied to this wounded cornea. The mice treated in this way were divided into four groups ( A), (B), (C) and (D) were classified as described below, the eyes of these mice were examined and the results were recorded photographically.

(A) 10 µl of a MEM solution containing 22.4 (ig 20 A / ml) was dropped on the cornea membrane of these mice 2 hours before application of HSV-1 (F strain). In addition, such treatment was daily from the day following HSV-1 (F strain) administration.

In addition, these mice received drops of a solution containing cortisone acetate (2 mg / kg mouse body weight) 5 hours before the injection of HSV-1 (F strain). Cortisone treatment was also continued daily.

(B) In the eyes of the mice, 10 (in a MEM solution containing 22.4 [ig 20 A / ml) was introduced 2 hours before the administration of HSV-1 (F strain). The treatment with 20 A was given to each continued the second day.

(C) These mice were given 30% of a solution containing cortisone acetate (2 mg kg body weight) as described in (A) above.

(D) 10 (µg of a MEM solution) was applied to the corneal membrane of these mice 2 hours before treatment with HSV-1 (F strain). In addition, from the day after treatment with HSV-1 (F strain ) performed a similar treatment with MEM solution every other day.

The condition of the eyes and the areas around the eyes of each mouse were examined with particular attention to inflammation and the closure of the eyes with pathological symptoms. The examination was carried out on the 4th, 7th or 12th day after the treatment. The mice of group (A) and group (B) showed much less pathological symptoms in the eyes and the surrounding areas than the mice in groups (C) and (D) who did not receive 20 A.

Furthermore, it can be concluded that the effectiveness of 20 A is not influenced by the use of 5 cortisone.

The following examples 20 to 22 illustrate the production of various antiviral drugs according to the invention.

Example 20

An oligodeoxynucleotide identical to 20 A, which was obtained in Example 3, is synthesized according to the triester method (using a triester of the corresponding nucleotides in the liquid phase). An isotonic MEM solution up to a final volume of 1 liter of injection solution is added to 2 g of 20 A prepared in this way. The solution is then filled into ampoules. This injection contains 2 mg 20 A / ml and is administered subcutaneously in a dose of 1 to 5 ml as close as possible to the infected part of the body if a symptom is observed.

Example 21

An oligodeoxynucleotide identical to 20 B, which was obtained in Example 4, is synthesized according to the Triester method. An isotonic MEM solution up to a final volume of 1 liter is added to 10 g of the 20 B synthesized in this way. This is a stock solution for a 1 percent eye drop preparation. This eye drop 30 fen preparation is given at a dose of 2 to 3 drops several times a day if a symptom is observed.

Example 22

An oligodeoxynucleotide identical to 20 A, which was obtained in Example 35, is synthesized according to the Triester method. 1 g of the 20 A synthesized in this way is ground to a fine powder and 999 g of purified cocoa butter are added to this fine powder. The mixture is kneaded in a water bath at 60 ° C. and 40 is then shaped into suppositories each weighing 2 g. Each suppositorium contains 2 mg of 20 A and is used according to the symptom.

The following Examples 23 and 24 illustrate the safety of the antiviral active substances according to the invention.

45

Example 23

No changes are observed in the test animals when 20 A is administered intraperitoneally to BALB / c mice in a quantity of 100 (ig / kg).

50

Example 24

As the test for confirming the effect described in the above example, the same test as that described in Example 55 of 19 was carried out, except that HSV-1 was not used. In this test, no difference was found between the group of test animals that had been given 20 A and the group that had not been given 20 A.

In view of the examples described above, it is obvious that the antiviral drugs according to the invention are safe with an effective dose of the oligo- and poly-deoxynucleotide.

The examples described below are intended to show 65 that an in vitro protein synthesis system is actually inhibited by hybridization of the (-) - strand 01-igodeoxynucleotide with the mRNA. These results support the theoretical background of this invention.

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Example 25 (Reference Example 1)

This example illustrates the inhibition of polyphenyl-alanine synthesis depending on poly U by oligode-oxyadenylic acid.

The reaction mixture (40) 1 contained 140 (poly U, 4C phenylalanine (105 cpm, 300 microcurie / micromol), E. coli extract, produced according to Nirenberg and Mathaei (Proc. Nat. Acad. Sei USA 47, page 1588 (1961)), 13 mM magnesium acetate, 1 mM ATP, 1 mM creatine phosphate, 1 jag creatine phosphokinase, 10 mM Tris-HCl (pH 7.5) and various amounts of oligodeoxyadenylic acid ml Eppendorf tubes were introduced and incubated for 30 minutes at 37 C. The amount of polyphenylalanine synthesized was determined by measuring the radioactivity insoluble in 95 ° C. hot, 10% trichloroacetic acid. The count was carried out in a liquid scintillation counter. Percent inhibition was calculated as a ratio to the polyphenylalanine synthesis value in the absence of oligodeoxyadenylic acid, and the results are summarized in Table VIII.

Table VIII

Table VIII (continued)

Inhibitor

Dose inhibition of poly

(Hg) Phenylalanine synthesis

Control '

poly-deoxyadenylic acid 50

oligo-deoxyadenylic acid 100 (10 nucleotides)

oligo-deoxyadenylic acid 100 (9 nucleotides)

0 98

99

Inhibitor

Dose inhibition of poly

(| ig) Phenylalanine synthesis oligo-deoxyadenylic acid 100 15

(8 nucleotides)

oligo-deoxyadenylic acid 100 0

(7 nucleotides)

Table VIII shows that the poly-deoxyadenylic acid that hybridizes with polyuridylic acid is a remarkably potent inhibitor of polyphenylalanine synthesis. i5 It can also be seen from this table that oligo-deoxyadenylic acid with a chain length of more than 9 nucleotides has a strongly inhibitory effect.

Example 26 20 (reference example 2)

The experiment described below is evidence that oligodeoxynucleotides can inhibit eukaryotic mRNA activity by forming hybrids with this mRNA. The reaction mixture 25 (30 (il) contains 4.2 mM calcium phosphate, 2 mM dimethyl-thiothreitol (DTT), 0.08 mM each amino acid (except methionine) 6 mM calcium acetate, 8 mM magnesium acetate, 4.5 ng spermidine, 0.1 (iCi 3:, S-methionine and 10 ml of a rabbit reticulocyte extract which was prepared according to Jackson and 30 Pelham (Europ. J. Biochem. 67, pages 247-256 (1976)). The inhibitor was dissolved in a 21 mM HE-PES solution and added to the reaction mixture as described in Table IX.

Table IX

Inhibitor

Dose (Hg)

% Inhibition of a-globin synthesis

% Inhibition of the β-globin synthesis

Control -

a-globin genomic DNA 0.06

β-globin genomic DNA 0.06

Calf thymus DNA 0.06

15 oligodeoxynucleotide of geno-0.06 mixer a-globin DNA

15-oligodeoxynucleotide of 0.06 M 13

0

60 0 10

0 0

65 10 0

As shown in Table IX, (-) - stranded a-globin DNA specifically inhibits a-globin synthesis, while (-) - stranded ß-globin DNA specifically inhibits ß-globin synthesis. No significant inhibition was observed with calf thymus DNA. From these findings it can be concluded that (-) - stranded DNA of a eukaryotic gene specifically inhibits the synthesis of the protein encoded by this gene. Oligodeoxynucleotides with a chain length of 15, which correspond to the NH2-terminal amino acid sequence of the a-globin, inhibited the synthesis 55 of a-globin. This observation indicates that single-stranded (-) - stranded DNA with a chain length of only 15 nucleotides is sufficient to specifically inhibit the synthesis of the corresponding protein. The control oligonucleotide (M 13 phage DNA with a chain length of 15 60 nucleotides) showed no significant inhibitory effect.

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4 sheets of drawings

Claims (8)

667 593 1. Medicament for the treatment of viral infections containing at least one oligonucleotide and / or polydeoxynucleotide consisting of 9-100 nucleotides in the form of a (-) - DNA strand which contains at least one DNA which completely or partially hybridizes with an mRNA generated during virus replication. Contains segment and a pharmaceutically acceptable excipient suitable for the treatment of viral infections. 2. Medicament according to claim 1 in a dosage form suitable for the treatment of RNA virus infections. 2nd PATENT CLAIMS 3. Medicament according to claim 1 in a suitable dosage form for the treatment of influenza virus infections. 4. Medicament according to claim 1 in a dosage form suitable for the treatment of DNA virus infections. 5. Medicament according to claim 1 in a dosage form suitable for the treatment of herpes simplex virus infections. 6. Medicament according to claim 1, containing a mixture of the oligo- and or polydeoxynucleotides. 7. A method for producing a medicament according to claim 1, characterized in that an oligo- or polydeoxynucleotide of 9-100 nucleotides in the form of a (-) - DNA strand is made from at least one fragment from the coding region of the Vi-rus genome isolated, which contains at least one DNA segment which hybridizes in whole or in part with an mRNA generated during virus multiplication and which mixes the oligo- or polydeoxynucleotide with a pharmaceutically acceptable excipient suitable for the treatment of virus infections. 8. A method for producing a medicament according to claim 1, characterized in that one synthesizes by chemical synthesis an oligo- or polydeoxynucleotide from 9-100 nucleotides in the form of a (-) - DNA strand, which at least partially or completely with a the virus replication generated mRNA hybridizing DNA segment and the oligo- or polydeoxynucleotide with a pharmaceutically acceptable excipient suitable for the treatment of virus infections.